This invention relates to microorganisms having mosquitocidal activity but lacking hemolytic activity. The invention further relates to recombinant DNA molecules encoding mosquitocidal polypeptides of the invention, to vectors containing the DNA molecules, and to microorganisms expressing the polypeptides.
Bacillus thuringiensis serovar israelensis produces crystalline inclusions during sporulation that are toxic to mosquito and blackfly larvae (15, 35). These inclusions are, when solubilized, cytolytic for various mammalian cells including erythrocytes (33). The crystals are composed of at least four polypeptides of 135 kDa, 125 kDa, 68 kDa, and 28 kDa. It has been shown by electron microscopic studies that these crystals are composite, and consist of three major inclusion types differentiated on the basis of electron opacity, size and shape. Purification and protein analysis of one of three types of component crystal revealed that it only contained the 68 kDa polypeptide (21). It was therefore suggested that the 28 kDa and 125-135 kDa polypeptides could be assembled separately in the two other inclusions (21).
The diversity of the polypeptides has complicated the identification of the protein(s) responsible for larvicidal activity. The major difficulty in identifying the toxic polypeptide(s) was the purification of the different polypeptides. One solution to this problem was the cloning of the different genes. It has previously been shown that the toxin genes of B. thuringiensis israelensis were located on a 72 MDa resident plasmid (16, 37). No copy of the toxin gene was found on the chromosomal DNA. Cloning experiments with the 72 MDa plasmid and analysis of the cloned products clearly indicated that the 135 kDa, 125 kDa, and 68 kDA were involved in the toxicity to mosquitoes, alone or in combination (reviewed in Federici et al. (13)).
There is still controversy concerning the activity of the 28 kDA protein. Although biochemical and cloning experiments revealed that this polypeptide is responsible for the in vitro cytolytic activity of the crystals (4, 7, 18, 20, 27, 40), its contribution to the mosquitocidal activity remains unclear. Earlier biochemical studies suggested that the 28 kDa polypeptide was not toxic to Aedes aegypti larvae (9, 18, 20, 30, 36). However, several groups found that the purified protein was toxic for this insect, although the LC.sub.50 observed was much higher than that obtained for the native, composite crystals (4, 10, 11, 22, 29). The cloning of the 28 kDa protein gene, now referred to as the cyta gene (19), on multicopy plasmids either in Escherichia coli (7, 27, 38, 40) or in Bacillus subtilis (41) did not resolve this controversy. One of the issues in the debate is the level of activity at which a polypeptide should be considered to be active. Moreover, if the 28 kDa protein acts synergistically with the 68 kDa and/or the 130 kDa polypeptides as suggested by Wu and Chang (42), Ibarra and Federici (21) and Chilcott and Ellar (10), the study of the cloned 28 kDa product alone does not reflect the involvement of this protein in the overall toxicity of the crystals.
There exists a need in the art for polypeptides having mosquitocidal activity and for recombinant vectors encoding the polypeptides and capable of expressing the polypeptides. Ideally, the microorganisms exhibiting mosquitocidal activity should be non-hemolytic.